An ion mobility spectrometer typically comprises an ionization source, a drift cell, and an ion detector. Examples of an ion detector include a faraday sampling plate or cup, an electron multiplier, or a mass spectrometer. Ion mobility spectrometry characterizes ions which are forced by an electric field to move a drift/buffer gas by measuring the ion's equilibrium drift velocity. When gaseous ions in the presence of the drift gas experience a constant electric field, they accelerate until the occurrence of a collision with a neutral atom or molecule within the drift gas. This acceleration and collision sequence is repeated continuously. Over time, this microscopic scenario averages the instantaneous velocities over the macroscopic dimensions of the drift tube resulting in the measurement of a constant ion velocity based upon ion size, charge and drift gas pressure. The ratio of the ion velocity to the magnitude of the electric field is defined as ion mobility. In other words, the ion drift velocity (vd) is proportional to the electric field strength (E), where the ion mobility K=vd/E is a function of ion volume/charge ratio. Thus IMS is a separation technique similar to mass spectrometry. IMS is generally known to have high sensitivity with moderate resolving power. Separation efficiency is compromised when “bands” of ions spread apart as opposed to arriving together at the end of the IM drift tube in a tight, well-defined spatial region.
The resolving power of an ion mobility cell increases as the square root of the voltage when a uniform (or quasi-uniform) electric field is imposed across an ion mobility cell. It would seem that there is not much freedom to increase the resolution; however, the situation may be improved if the ion drift in a gas flow is considered. Ions move against a counterflow of gas only if the field is stronger than a certain value specific for the mobility of the ions. Ions with lower mobility may be stationary or even move in the negative direction (with the gas flow). Therefore, better ion separation can be expected where the time of the mobility separation can be chosen suitable for specific applications and compatible with the time diagram of the ion detector operation. The problem is how to efficiently organize ion mobility separation using gas counter-flow. Most often ion mobility separation is used with ion sources working under elevated pressure and the source pressure is often used when these ions are introduced into a mobility cell. There may be no gas counter-flow in such an application. On the other hand, drift gas counter-flow is inevitable when IMS is used for analysis of ions created in high vacuum ion sources such as a secondary ion source where secondary ions are created from a surface maintained in high vacuum and must then be moved against a counter-flow of gas into the ion mobility spectrometer. The main problem then is how to overcome the strong counter-flow and preserve ion throughput. It is quite natural to use a time varying electric field to gradually move ions from a pulsed ion formation region against the gas flow and into the IMS. Small ions need a relatively small field to overcome the gas flow without decomposing whereas larger ions can come to the entrance orifice later under the action of a stronger field. At the time of application of the larger field necessary to move the heavier ions, small ions are already inside the mobility cell and are not subjected to the strong field which would otherwise cause their fragmentation. Some separation of ions in addition to the usual mobility separation is achieved in this case, however, it is often rather small, because of the diffusion broadening during the initial ion cloud formation.
The combination of an ion mobility spectrometer (IMS) with a mass spectrometer (MS) is well known in the art. In 1961, Barnes et al. were among the first to combine these two separation methods. Such instruments allow for separation and analysis of ions according to both their mobility and mass, which is often referred to as two-dimensional separation or two-dimensional analysis. Young et al. realized that a time-of-flight mass spectrometer (TOFMS) and specifically an orthogonal TOFMS is the most preferred mass spectrometer type to be used in such combination because of its ability to detect simultaneously and very rapidly (e.g. with high scan rate) all masses emerging from the mobility spectrometer. The combination of a mobility spectrometer with a TOFMS is referred to as a Mobility-TOFMS. This prior art instrument comprised means for ion generation, a mobility drift cell, a TOFMS, and a small orifice for ion transmission from the mobility cell to the TOFMS.
In 2003, Loboda (U.S. Pat. No. 6,630,662) described a method for improving ion mobility separation by balancing ion drift motions provided by the influence of DC electric field and counter-flow of the gas. Using this balance, ions are at first accumulated inside an ion guide, preferably an RF-ion guide, and then, by changing the electric field or gas flow, the ions are gradually eluted from the ion guide to the mass spectrometer. Such type of ion accumulation is restricted to collecting relatively small number of ions due to space-charge effects. It also has some limitation in ion mass-to-charge (m/z) range because RF-focusing for a given RF-voltage has decreasing efficiency for larger mass ions which cannot be improved by increasing the RF-voltage due to the possibility of creating a glow discharge at the relatively high gas pressure inside the RF multipole. Unfortunately at lower pressure the influence of the gas flow on ions is less and the diffusion of the ions increase so trapping and separation of larger ions could be compromised. The time of ion accumulation and their storage in the RF-ion guide cannot be too long, otherwise ions would be partially lost due to diffusion into rods or walls confining the gas flow. For at least these reasons, this method has significant resolving power limitations. The instrumental improvements disclosed below eliminate these drawbacks.
Use of MS as a detector enables separation based on mass-to-charge (m/z) ratio after the separation based on ion mobility. Shoff and Harden pioneered the use of Mobility-MS in a mode similar to tandem mass spectrometry (MS/MS). In this mode, the mobility spectrometer is used to isolate a parent ion and the mass spectrometer is used for the analysis of fragment ions (also called daughter ions), which are produced by fragmentation of parent ions. Below this specific technique of operating a Mobility-MS is referred to as Mobility/MS, or as Mobility-TOF if the mass spectrometer is a TOFMS-type instrument. Other prior art instruments and methods using sequential IMS/MS analysis have been described (see, e.g., McKight, et al. Phys. Rev., 1967, 164, 62; Young, et al., J. Chem. Phys., 1970, 53, 4295; U.S. Pat. Nos. 5,905,258 and 6,323,482 of Clemmer et al.; PCT WO 00/08456 of Guevremont) but none combine the instrumental improvements disclosed here. When coupled with soft ionization techniques and the sensitivity improvements obtained through the use of the drift cell systems disclosed herein, the IMS/MS systems and corresponding analytical methods of the present invention offer significant analytical advantages over the prior art, particularly for the analysis of macromolecular species, such as biomolecules.
One challenge when building a Ion Mobility-MS system is to achieve high ion transmission from the mobility region into the MS region. It is at this interface that earlier uses of linear fields appear incongruous with the goal of maximizing ion throughput across the IMS/MS interface. The mobility section operates at typical pressures between 1 mTorr and 1000 Torr whereas the MS typically operates at pressures below 10−4 Torr. In order to maintain this difference in pressure it is necessary to restrict the cross-section of the exit orifice of the IM drift cell so that the region between the IM and the MS can be differentially pumped. Typically this orifice cross section is well below 1 mm2. Hence it is desirable to focus the ions into a narrow beam before they reach the interface. Another essential property of the ion beam coming into an oTOFMS is the beam divergence, or the kinetic energy of ion motion in the plane orthogonal to the direction of their travel. This is the main factor responsible for the quality of mass spectra obtained in the orthogonal TOFMS. It is a subject of our two co-pending U.S. patent applications: U.S. application Ser. No. 11/441,766 filed May 26, 2006; and U.S. application Ser. No. 11/441,768 filed May 26, 2006 to achieve good ion beam properties by using ion cooling in supersonic adiabatic gas flow. Both of these applications are incorporated by reference as though fully set out herein.
Tandem mass spectrometry techniques typically involve the detection of ions that have undergone some structural change(s) in a mass spectrometer. Frequently, this change involves dissociating or fragmenting a selected precursor or parent ion and recording the mass spectrum of the resultant daughter fragment ions. The information in the fragment ion mass spectrum is often a useful aid in elucidating the structure of the precursor or parent ion. The general approach used to obtain a mass spectrometry/mass spectrometry (MS/MS or MS2) spectrum is to isolate a selected precursor or parent ion with a suitable m/z analyzer, subject the precursor or parent ion to an energy source to effect the dissociation (e.g. energetic collisions with a neutral gas in order to induce dissociation), and finally to mass analyze the fragment or daughter ions in order to generate a mass spectrum. An additional stage of isolation, fragmentation and mass analysis can be applied to the MS/MS scheme outlined above, giving MS/MS/MS or MS3. This additional stage can be quite useful to elucidate dissociation pathways, particularly if the MS2 spectrum is very rich in fragment ion peaks or is dominated by primary fragment ions with little structural information. MS3 offers the opportunity to break down the primary fragment ions and generate additional or secondary fragment ions that often yield the information of interest. The technique can be carried out n times to provide an MSn spectrum.
Ions are typically fragmented or dissociated in some form of a collision cell where the ions are caused to collide with an inert gas. Dissociation is induced usually either because the ions are injected into the cell with a high axial energy or by application of an external excitation. See, for example, WIPO publication WO 00/33350 dated Jun. 8, 2000 by Douglas et al. Douglas discloses a triple quadrupole mass spectrometer wherein the middle quadrupole is configured as a relatively high pressure collision cell in which ions are trapped. This offers the opportunity to both isolate and fragment a chosen ion using resonant excitation techniques. The problem with the Douglas system is that the ability to isolate and fragment a specific ion within the collision cell is relatively low. To compensate for this, Douglas uses the first quadrupole as a mass filter to provide high resolution in the selection of precursor ions, which enables an MS2 spectrum to be recorded with relatively high accuracy. However, to produce an MS3 (or higher) spectrum, isolation and fragmentation must be carried out in the limited-resolution collision cell.
A three-dimensional ion trap (3-D IT) is one of the most flexible devices for MS-MS and multi-step (MSn) analysis. The basic operation principle of the quadrupole ion trap mass spectrometer is well-known (for example, refer to U.S. Pat. No. 2,939,952, Paul et al., June, 1960). This trap is composed of a ring electrode and two end cap electrodes of special shape to create a quadrupolar distribution of potential. Radio frequency (RF) and DC offset electric potentials are applied between electrodes and cause ions to oscillate within the trap. By appropriately selecting voltage parameters, ions of a specific mass/charge ratio can be made to have stable or unstable trajectories. In another implementation an additional (auxiliary) AC voltage is applied to the end-caps to induce resonant excitation of selected ions either for the purpose of ejecting the selected ions or for the purpose of inducing collisional dissociation. The 3-D ion trap is capable of single step mass spectrometric analysis. In such analysis ions are injected into the trap (or generated within the trap), confined to the center of trap because of low energy collisions with an inert gas such as helium (typically at 1 mtorr pressure) and then sequentially ejected through the apertures in the end cap electrodes onto an external detector by raising the amplitude of the RF field. The same device could be used for a multi-step, i.e. MSn, analysis—U.S. reissued Pat. No. 34,000, Syka et al., July, 1992. The ion trap isolates ions in a m/z window by rejecting other components, then fragments these isolated ions by AC excitation, then isolates resulting ion fragments in a m/z window and repeats such sequence (MSn operation) in a single cell. At the end of the sequence ions are resonantly ejected to acquire the mass spectrum of N-th generation fragments. The 3-D IT is vulnerable to sensitivity losses due to ion rejection and instability losses at the time of ion selection and fragmentation.
Another version of this technique is provided by hybrid instruments combining quadrupoles with time of flight analyzers (Q-TOF) where the second quadrupole mass spectrometer (in a triple quadrupole systems) is replaced by an orthogonal time of flight spectrometer (o-TOF). The o-TOF back end allows observation of all fragment ions at once and the acquisition of secondary spectra at high resolution and mass accuracy. The Q-TOF has huge advantages in cases where the full mass range of daughter ions is required, for example, for peptide sequencing, the Q-TOF strongly surpasses the performance of the triple quadrupole. However, the Q-TOF suffers a 10 to a 100 fold loss in sensitivity as compared to a single quadrupole mass filter operating in selected reaction monitoring mode (monitoring single m/z). For the same reason the sensitivity of the Q-TOF is lower in the mode of “parent scan” where, again, the second MS is used to monitor a single m/z. Usually, only one step of MS/MS analysis is possible for such types of instruments. Recently, the quadrupole has been replaced by a linear ion trap (LIT)—U.S. Pat. No. 6,020,586, Dresch et al., February, 2000. The quadrupole with electrostatic “plugs” is capable of trapping ions for long periods of time. The quadrupole field structure allows one to apply an arsenal of separation and excitation methods, developed in 3-D ion trap technology, combined with easy introduction and ejection of the ion beam out of the LIT. The LIT eliminates ion losses at selection and also can operate at poor vacuum conditions which reduces requirements on the pumping system. However, a limited resolution of ion selection, R<200, has been demonstrated thus far. A method for improving the sensitivity in LIT is described in U.S. Pat. No. 6,507,019, Chernushevich et al., January, 2003. According to this method, a voltage on the outlet of the collision chamber is controlled in synchronization with the timing of applying an acceleration voltage in a time-of-flight mass spectrometer thereby improving the sensitivity for ions in a specified range of m/z.
Fourier transform ion cyclotron resonance mass spectrometry (FTMS) currently provides the most accurate measurement of ion mass to charge ratios with a demonstrated resolution in excess of 100,000. In FTMS, ions are either injected from outside the cell or created inside the cell and confined in the cell by a combination of static magnetic and electric fields (Penning trap). The static magnetic and electric field define the mass dependent frequency of cyclotron motion. This motion is excited by an oscillating electric potential. After a short period the applied field is turned off. Amplifying and recording weak voltages induced on the cell plates by the ion's motion detects the frequency of ion motion and, thus, the m/z of the ion. Ions are selectively isolated or dissociated by varying the magnitude and frequency of the applied transverse RF electric potential and the background neutral gas pressure. Repeated sequences of ion isolation and fragmentation (MSn operation) can be performed in a single cell. The possibility of kinetic measurements of ion dissociation using controlled black body heating of ions so called BIRD technique is another unique property of these type instruments. However, it may be considered as rather basic research tool than analytically useful approach. An FTMS is a “bulky” device occupying a large footprint and is also expensive due to the costs of the magnetic field. Moreover, an FTMS exhibits poor ion retention in MSn operation (relative to the 3-D ion trap).
Use of an AC field for selected ion rotation within a gas filled RFQ axis has been described (Raznikov, et. al., RCM, 15, 1912-1921, 2001). Such ion motion was used for ion heating and fragmentation of selected ions by collisions with buffer gas. It was demonstrated that resolving power (FWHM) was near 80 for mass selecting the parent ions (within the m/z range 500-1000) which could then be selectively decomposed under N2 pressure close to 20 mTorr. Kinetic measurements are enhanced when using this technique (Soulimenkov, et. al., Europian Journal of Mass Spectrometry 8, 99-105, 2002). Rotating ions around the axis of a gas filled RFQ is one of the particular cases of two-dimensional motion in an axially symmetric quadratic potential well provided by a quadrupolar RF-field. This motion is influenced by harmonic voltages applied to adjacent RFQ rods with phase shift π/2. Upon comparison with other types of ion oscillations in which the ion distance from RFQ axis varies over a wide range, ion rotation has some advantages conferred by the properties of classic harmonic motion. For simple ion oscillations (for dipole or quadrupole excitations), the ion velocity is not constant and the ions come to rest at the maximum deviation from the axis, whereas for rotating ion motion the velocity is almost constant. There are two advantages to having a constant average kinetic energy for a given maximum rotational orbit deviation from the axis. First, as a consequence of the constant ion velocity the conditions for observation of fragment ions, especially including low m/z values, can be considerably better known and controlled than merely using a quadrupolar field alone. Otherwise, in order to achieve the same average ion internal energy in the case of quadrupolar oscillations, it is necessary to have either larger maximum deviations of ions from the RFQ axis or to have a stronger ion focusing to the axis which then demands larger amplitude or lower frequency of the RF-voltage. The second advantage is that ion rotation gives more control of ion heating and decomposition under the usual conditions employed in the RFQ where the gas density is nearly uniform.
In addition to ICR mass spectrometry, other applications of ion rotation are found in DC traps like the Orbitrap instrument (disclosed by Makarov in 1999, U.S. Pat. No. 5,886,346) and FTMS based on ion rotation in a linear RF multipole ion trap (described by Park in 2004, U.S. Pat. No. 6,784,421). In all these instruments ions rotate freely in high vacuum after a short voltage pulse starts the ion motion. The linear RF multipole ion trap is the closest prior art to ion rotation in a gas filled RFQ. In both types of instrument the resolving power restrictions are dependent on the accuracy of manufacturing of RF ion guide or linear trap. The Orbitrap arguably has some important advantages over these techniques, however, it is limited to analyzing ions of only one sign while both ICR and ion rotation in linear multipole ion traps allow simultaneous measurement of ions of both signs. All these techniques may provide extremely high resolution for moderate to small ions. For larger ions mass resolving power decreases rapidly for two main reasons: large ions rotate at a lower frequency than smaller ions and larger m/z are more prone to oscillations and thus have shorter time periods of uninterrupted coherent motion due to the increased probability of impact with residual gas resulting from their larger collision cross section. Moreover, inserting large ions into the DC trap portion of the Orbitrap has a further limitation since these ions may have a large probability to collide with gas in the storage quadrupole during their acceleration to the necessary relatively high energy for insertion (about 1 kV). Thus, during insertion of these larger ions into the Orbitrap, a significant portion of the large ion flux may be lost due to dissociation broadening of their energy distribution away from an optimal insertion energy at the moment of their capture in the DC trap. These instruments demand long measurement times (up to few seconds) and slightly more time for ion preliminary accumulation. Thus, they are not suitable for many types of ion mobility measurements since all mobility resolution will be lost during such ion accumulation as will any information about the average velocity of the incoming ions. Also, most often mass spectrometric techniques provide only semi-quantitative information about ion transformations. For real kinetic measurements it is necessary to have very narrowly defined energy distributions (or temperatures) of ions and of neutral reactant components combined with precise measurement of reaction times. The only methods capable of manipulating these parameters use a direct heating of the reaction zone (like BIRD, for example). However, external heating of some parts of the instrument may result in significant experimental problems. The method of selective ion heating with a resonant rotating field also has a limited capability to specify both the temperature of the ions of interest and the time at which heating occurs since the temperature of rotating ions for a given field strength is dependent on ion mobility. Unfortunately, it is necessary to know this temperature under conditions when ions begin to decompose yet this temperature can only be effectively measured when ions have not yet decomposed. Furthermore, to have high selectivity with resonant ion rotation the buffer gas pressure should be small which then requires an inordinately long time for the selected ions to reach the desired steady state temperature. The same limitation occurs at the end of an RFQ when the rotating field is switched off to allow focusing the ions for subsequent measurement in the TOFMS.
Two dimensional, 2-D PAGE polyacrylamide gel electrophoresis is a popular and currently preferable technique for protein separation (Anderson N. G., Anderson N. L., Electrophoresis 1996; 17, 443453). Proteins are subjected at first to isoelectric focusing (IEF) in an immobilized pH gradient in the gel plate to separate proteins according to their charging abilities (pI values), a step which typically takes about 6-8 hours. Then, the IEF gel is placed on top of a gradient gel and is subjected to electrophoretic separation in the presence of SDS (sodium dodecyl sulphate). In SDS-PAGE, proteins are denatured and dissolved in a SDS buffer, negatively charged SDS molecules bind to the protein, with more molecules binding to larger proteins. On application of an electric field, proteins migrate in a polyactylamide gel according to their charge connected with size or mass. The electric field is switched off to immobilize the proteins within the gel. The separated proteins are stained for visualization, bands of interest are excised and digested with protease followed by mass spectrometry measurements for protein identification (Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Boucherie, H., Mann, M., Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445; Jensen, O. N., Larsen, M. R., Roepstorff, P., PROTEINS 1998, 74-89 Suppl. 2). 2-D PAGE is the current technology of choice for large scale proteomics analysis because 2-D PAGE at the moment is the highest resolution method for protein separation and the spots of proteins in the 2-D map are related to the properties of proteins, namely isoelectric point in the first dimension and the molecular mass in the second dimension. Therefore, the positions of proteins in 2-D map correspond to their chemical and physical properties. These properties can be used to identify and characterize the proteins. 2-D PAGE has been used to analyze human plasma proteins, and the pI and molecular weight of proteins can be used for detection and diagnosis of diseases in clinical analysis (Rasmussen, R K., Ji, H., Eddes, J. S., Moritz, R L., Reid, G. E., Simpson, R. J., Dorow, D. S., Electrophoresis 1997, 18, 588-598). However, 2-D PAGE is a time consuming procedure which is difficult to automate, it also suffers from limitations in sensitivity and dynamic range of detection. Virtual 2-D gel electrophoresis has recently been developed (Ogorzalek-Loo, R. R., Cavalcoli, J. D., VaiBogelen, R A., Mitchell, C., Loo, J. A., Moldover, B., Andrews, P. C., Anal. Chem. 2001, 73, 40634070), where mass spectrometry replaces the size-based separation of SDS-PAGE in the second dimension. It has been shown that this technology is more sensitive than 2-D PAGE. However, the first dimension of separation is still performed in a polyacrylamide gel, limiting the potential for high throughput analysis. Capillary isoelectric focusing (CIEF) is an equilibrium-based method of separation that depends on a pH gradient created by carrier ampholyte. Proteins move under an electric field to their pI points where they carry zero average charge and are focused. Therefore, separation and concentration occur at the same time. The concentration of proteins at the focused zone can be increased by 100-500 times relative to the starting solution because the same protein in the whole capillary is focused on a single spot. Single point detection techniques, such as laser induced fluorescence and ESI-MS, have been employed to detect the separated proteins after CIEF. Focused protein zones need to be mobilized in order to pass through the detection point at the end of the tube (Rodriguez, R., Zhu, M., Wehr, T., J Chromatogr. A 1997, 772, 145-160). The problem of interfacing CIEF with MALDI-MS is also because the focused protein zone inside the capillary cannot be reached directly. Therefore, the contents of the capillary need to be moved out of the capillary and deposited into an appropriate surface for subsequent MALDI-MS analysis. This mobilization step degrades the resolution, increases the analysis time, and distorts the pH gradient. Hence, the result reproducibility is poor.
All of the above-referenced U.S. patents and published U.S. patent applications are incorporated by reference as though fully described herein.
Although much of the prior art has resulted in improvements in ion focusing, separation and in ion throughput from ion source to the mobility cell (and to the mass spectrometer in tandem instruments), there is room for additional improvement in all these areas. The inventors describe herein a concept and design of a new type of interface of an ion mobility cell with an orthogonal injection time-of-flight mass spectrometer (TOFMS) based on significant cooling of the ion beam in an adiabatic supersonic gas flow and its focusing by radio-frequency quadrupole or multipole ion guide with additional DC and AC rotating fields which result in variety of instrumental embodiments to provide improved ion production from investigated samples, their separation and measurements. The modified ion rotational trapping, manipulation, and measurement technique disclosed in the present invention is free from the limitations of the prior art as the mobility cross-sections of any ion of interest will have always been measured prior to its introduction into the RFM. Furthermore, the drift velocities of these ions and those for the components of the buffer gas and their divergences (which can be related to their temperatures) are essential for obtaining quantitative kinetic information and these properties can be uniquely measured by innovative use of multianode position sensitive detector combined with a multi-channel data recording system in a TOFMS. One of the aims of the present invention is to perform isoelectric separation of biomolecules in the gas phase and reduce the time of conventional procedure in gel separation from 6-8 hours to few seconds or minutes depending on the problem to be solved. Also the problems of interfacing of TOFMS with separating devices in liquid phase would be avoided in this case.
Time-of-flight instruments seem to be more suitable than prior art instruments for measurement of ions coming from an RF ion guide with supersonic gas flow, especially when investigation of large bioions is the main analytical problem. Divergence of large ions in the supersonic gas flow and their final focusing in an RF ion guide technically can be done significantly better than those for relatively small ions. Therefore, it is quite realistic to provide better resolving power for large ions in TOF measurements than for those ions with less mass and smaller size. Furthermore, the resolution of multicharged ions varies proportionally to the square root of the charge number at least for a linear oTOFMS. Moreover, the expected peak shape, at least for a linear gridless TOFMS, would be close to Gaussian which is significantly better then Lorentzian peak shapes typical for FTMS instruments. This is an especially useful advantage when measuring a small peak adjacent to a large one. Multianode data acquisition allows not only to increase the dynamic range of the ion detection but also to measure ion velocities and their divergences thus providing direct estimations of their temperatures. These possibilities are important for quantitative kinetic measurements.